人降钙素基因在大肠杆菌中的克隆与表达

The gene cloning and expression of human calcitonin in Escherichia coli

目的：利用大肠杆菌表达具有生物活性的人降钙素（ｈＣＴ）。方法：以人类基因组ＤＮＡ为模板，经ＰＣＲ扩增获取ｈＣＴ基因片段，测序分析克隆到ｐＵＣ１９载体中的ＰＣＲ产物，确定得到两种ＣＴ基因序列，其中１＃与文献报道完全一致，２＃序列中含有（Ｇ６４／Ａ）点突变。采用带有热诱导启动子的高效表达载体ｐＢＶ２２０，对所获得的基因进行了表达。并利用大鼠降血钙实验，对表达产物的活性进行了检测。结果：所获得的ｒｈＣＴ有降血钙的活性，正常基因的表达产物活性略高于突变基因的产物。结论：利用大肠杆菌可表达有生物活性的ｈＣＴ。

Objective: To express the biological active human calcitonin (hCT) in E.coli. Methods: The human genomic DNA was chosen as the template to obtain the CT gene through PCR. The result of the sequencing of the PCR products which had been cloned into the pUC19 vector showed that 1# gene had the same sequence as that had been published, while 2#gene had a single mutation at the site of 64th base pair(A→G). Both genes were then cloned into the expression vector pBV220, which has a strong and temperaturedependent promoter. Biological activity of the recombinant hCT was tested for the hypocalcemic effect on rat. Result: The two kinds of the rhCT had hypocalcemic activity, while recombinant product of the wide type gene had a little higher activity. Conclusion: The hCT with high biological activity can be expressed in E.coli.